The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we use a unique approach in which the centrosome (labeled with fluorescent proteins) is ablated by a laser microbeam. This approach allows us to engineer cells that lack the centrosome, and compare their behavior with genetically identical cells that possess this organelle. Further, we combine laser microsurgery with inactivation of individual proteins by siRNA and cell-permeable compounds. This synergistic methodology provides new information unobtainable by other means. Our current research focuses on two areas. First, we are testing the hypothesis that improper balance between the contributions of centrosomal and centrosome- independent mechanisms during the assembly of mitotic spindle assembly leads to chromosomal instability. We follow the behavior of individual chromosomes in human cancer cells by high-resolution microscopy and investigate how this behavior changes when the centrosomes are laser ablated (Aim 1). We also study how chromosome behavior changes in response to perturbations of key molecules implicated in the centrosome- independent microtubule organization (TPX2 and HURP) or in the correction of erroneous chromosome orientations (Aurora B and kinesin 13s) (Aim 2). Finally, we use laser microsurgery, cell-permeable chemical inhibitors, and siRNA to study molecular and structural requirements for 'centriole disengagement', a key event in the centrosome cycle that must be properly controlled to prevent repetitive re-duplication of centrioles in the same cell cycle (Aim 3). Our observations suggest that daughter centrioles cannot disengage until they reach a certain level of maturation. Thus, control of centriole disengagement involves a previously overlooked mechanism that is intrinsic to the centrosome. We currently investigate molecular mechanisms responsible for procentriole maturation.